ABSTRACT Cartilage is a soft tissue involved in bone lengthening, fracture repair and painless joint motion. Damage to skeletal cartilages can hinder bone development and cause painful joint diseases like osteoarthritis (OA), which is a leading cause of disability and hospital visits in the USA and around the world. This project will explore how the enzymes Phlpp1 and Phlpp2 regulate the formation, maintenance and regeneration of skeletal cartilage. Phlpp1 and Phlpp2 are intracellular protein phosphatases that control chondrocyte anabolism by post-translationally modifying protein substrates (e.g., AKT, PKC?). Articular chondrocytes from OA patients express abnormally high levels of Phlpp1 and Phlpp2. Global deletion of Phlpp1 increases chondrocyte proliferation and matrix synthesis, and reduces injury-induced OA while stimulating the expression of cartilage growth factors and receptors (Fgf18, Tgf? and Pthr1). Phlpp1 or Phlpp2 depletion also induces the expression of small proteoglycans (Prg4, Dcn, Spp1) that improve cartilage tissue integrity and joint lubrication. In a preclinical animal model of post-traumatic OA, a single intra-articular injection of a small molecule Phlpp inhibitor temporarily reduced cartilage loss and pain associated with a meniscal injury without adverse events for five weeks. These Phlpp inhibitors also promoted chondrocyte proliferation and production of matrix genes. Thus Phlpp inhibition is a new therapeutic strategy for OA, but additional knowledge of how Phlpp1 and Phlpp2 regulate signaling pathways and promote chondrocyte anabolism is necessary to maximize the therapeutic potential of Phlpp inhibitors. The overall goals of this project are to elucidate novel molecular and cellular mechanisms of Phlpp1 and Phlpp2 action during endochondral development and post-traumatic OA pathogenesis, and to define the specificity of Phlpp inhibitors in injured articulating joints. The specific aims are to: 1) define the roles of Phlpp1 and Phlpp2 in proliferation and maturation of collagen 2-positive cells during skeletal development; 2) determine the molecular functions of Phlpp1 and Phlpp2 in adult aggrecan-expressing cells following a joint injury; 3) test the specificity of Phlpp inhibitors to Phlpp1 and Phlpp2 and improve their effectiveness in post-traumatic OA models; and 4) elucidate the contribution of GIRK channels to the integration of Phlpp1 and Phlpp2 with anabolic signaling events.